Genetic variations in sites of affinity
between FVIII and LRP1 are not
associated with high FVIII levels in venous
thromboembolism
Luis F. Bittar, Lucia H. Siqueira, Fernanda A. Orsi, Erich V. De Paula & Joyce M. Annichino-Bizzacchi
Hematology and Hemotherapy Center, University of Campinas, Campinas, SP, Brazil.Increased factor VIII (FVIII) levels are a prevalent and independent risk factor for venous
thromboembolism (VTE). The low density lipoprotein receptor-related protein 1 (LRP1) has been
associated with FVIII catabolism. After a median of 10 years of the first thrombotic episode, we evaluated
FVIII activity levels in 75 patients with VTE and high FVIII levels and in 74 healthy controls. Subsequently,
we evaluated the regions of
F8 and LRP1 genes coding sites of affinity between these proteins, with the
objective of determining genetic alterations associated with plasma FVIII levels. After a median time of 10
years after the VTE episode, FVIII levels were significantly higher in patients when compared to controls
(158.6 IU/dL vs. 125.8 IU/dL; P # 0.001]. Despite the fact that we found 14 genetic variations in
F8 and
LRP1 genes, no relationship was found between FVIII levels with these variations. We demonstrated a
persistent increase of FVIII levels in patients with VTE, but in a much lower magnitude after 10 years when
compared to 3-years after the episode. Moreover, we observed no relationship of genetic variations in the
gene regions coding affinity sites between LRP1 and FVIII with FVIII levels.
V
enous thromboembolism (VTE) is a multifactorial disease, and increased levels of coagulation factor VIII
(FVIII) have been established as a prevalent and independent risk factor for the first episode and recurrent
VTE
1–4. We previously demonstrated that increased levels of FVIII are associated with VTE in Brazilian
patients
5. The main determinants of FVIII in plasma are von Willebrand factor (VWF) and ABO blood group
6–7.
Although FVIII is an acute phase protein, results from previous studies suggested that increased FVIII levels in
VTE patients are independent of an acute reaction
8–11and a familial clustering was observed
12–14. Analysis of
FVIII gene showed several polymorphisms, but without any correlation with elevated FVIII levels
15–18.
The low density lipoprotein receptor-related protein 1 (LRP1), also known as a2-macrogobulin receptor or
CD91 is a large (600 kDa) multifunctional receptor which belongs to the low-density lipoprotein receptor family
of endocytic receptors. LRP1 is involved in clearance of many different ligands, including FVIII
19. The first
evidence of the role of LRP1 in clearance of FVIII was simultaneously described by two studies. The authors
showed by surface plasmon resonance analysis that FVIII binds to LRP1 with a moderate affinity. Moreover,
assays in cell culture of fibroblasts and in vivo animal model showed that radioactive FVIII (
I-125FVIII) specifically
binds to LRP1 mediating the internalization and subsequent degradation of FVIII, indicating that LRP1 plays an
important role in FVIII clearance
20,21. Bovenschen et al
22confirmed the role of LRP1 in the clearance of FVIII
employing an in vivo LRP1-deficient mouse model. Although several studies investigated the correlation between
polymorphisms in LRP1 gene and plasma levels of FVIII and VTE
14,23–26, only C663T polymorphism showed
association with elevated FVIII levels and VTE risk
25.
The ligand-binding sites within the extracellular domain of LRP1 are composed by four clusters of
complement-type repeats (CRs), and each CR is formed by approximately 40 amino acids. Clusters II and IV
were shown to be responsible for the binding of majority of LRP1 ligands, including FVIII. The LRP1 cluster II
consists of 8 CRs, and it was demonstrated that FVIII has affinity for the first six CRs of cluster II
27–29. Within the
FVIII molecule, two high-affinity binding sites for LRP1 have been identified: the region 484–509 of the A2
domain and the region 1811–1818 of the A3 domain; and a low-affinity site in the region 2303–2333 of the C2
domain
30–33.
SUBJECT AREAS:
DNA SEQUENCING THROMBOEMBOLISM GENETICS RESEARCHReceived
3 November 2014
Accepted
25 February 2015
Published
18 March 2015
Correspondence and requests for materials should be addressed to L.F.B. (lfbsckayer@ hotmail.com)Therefore, genetic variations in these regions could potentially
affect FVIII clearance, thereby influencing the plasma FVIII levels.
Herein, we investigated the presence of genetic variations in gene
regions coding sites of affinity between FVIII and LRP1 in patients
with history of VTE and persistently elevated FVIII levels.
Results
Median age of the 75 patients (22 male e 53 female) was 47 years, and
median time after the fist VTE episode was 10 years (range 8–15).
The control group consisted of 74 participants (28 male and 46
female) with a median age of 45 years. There was no significant
difference between patients and controls related to gender, age,
and ABO blood group (Table 1).
Venous thromboembolism was spontaneous in 28 (37.3%)
patients. In the remaining 47 (62.7%) patients, associated risk factors
were surgery (8.0%), immobilization (17.3%), oral contraceptive use
(21.3%), pregnancy and puerperium (12.0%), among other less
pre-valent causes (4.1%). The sites of VTE were: lower limbs (88,1%),
splenic-portal circulation (7,4%) and upper extremities (4,5%). Four
(5,3%) patients with venous thrombosis of the lower limbs also
pre-sented pulmonary embolism.
Table 1 shows that in the first assessment, with a median time of
the first VTE episode of 3 years (range 1–8), FVIII levels was
232.4 IU/dL, and significantly higher than in controls (126.9 IU/
dL; P # 0.001). In the second assessment, with a median time of
the first VTE episode of 10 years (range 8–15), FVIII levels was still
significantly higher in patients (158.6 IU/dL) than in controls
(125.8 IU/dL; P # 0.001), although with a lower magnitude.
In the first assessment, all 75 patients included in this study had
FVIII above the P90 (200.0 IU/dL) since thus was a prior inclusion
criteria for this study. During the second analysis, the P90 was
170.3 IU/dL, and only 26 patients (34.7%) remained above this
per-centile. Therefore, we observed a significant decrease in the number
of patients with increased FVIII levels in the second analysis (P #
0.001). CRP levels in patients with VTE were between normal values
(0.11 mg/dL) but significantly lower when compared to the first
measure with 3-year median after venous thromboembolism episode
(0.21 mg/dL; P 5 0.003).
We identified 14 genetic variations, including 12 in LRP1 gene and
2 in F8 gene. Molecular alterations previously described were
clas-sified as mutation (allelic prevalence , 1%) or polymorphism (allelic
prevalence $ 1%) according to their prevalence in the population of
NCBI database, composed of 1000 healthy individuals (2000 alleles).
Novel genetic variations were classified according to their prevalence
in the control group of the study, thus, two were classified as
poly-morphisms and twelve as mutations. Of the 12 mutations identified,
three had not yet been described. Despite the identification of several
genetic alterations, no significant difference was observed in their
prevalence between patients and controls, with no association with
VTE history (Table 2).
The comparison of plasma FVIII levels according to genotypes of
LRP1 polymorphisms in the VTE patients group did not show
stat-istical differences (Table 3).
Discussion
The mechanisms underlying the elevation of FVIII levels in VTE
patients are not totally established. The main determinants of
FVIII in plasma are the von Willebrand factor (VWF) level and
the ABO blood group. Moreover, age and ethnicity also have an
important role in the regulation of FVIII levels. Immediately after
its release into the circulation, FVIII is caught into a tight
non-cova-lent complex with the VWF. The VWF acts as a natural carrier of
Table 1 | Baseline Characteristics of Patients with Venous Thromboembolism and Control Group
VTE Patients (N575) Controls (N574) P*
Age - Median (Interquartile Range) 47 (40–55) 45 (36–51) 0.28
Gender - Female/Male (%) 70.7%/29.3% 62.2%/37.8% 0.29
ABO Blood Group - Non-O/O (%) 68.0%/32.0% 67.6%/32.4% 1.00
FVIII - First Assessment (2004) [IU/dL](Interquartile) 232.4 (216.2–286.1) 126.9 (98.8–145.8) #0.001 FVIII - Second Assessment (2011) [IU/dL](Interquartile) 158.6 (148.5–171.0) 125.8 (93.4–148.6) #0.001
This table shows baseline characteristics of VTE patients and controls.
*P values were calculated by the Fisher’s exact test for categorical variables and Mann-Whitney test for continuous variables. Abbreviations: FVIII 5 factor VIII; VTE 5 venous thromboembolism.
Table 2 | Genetic variations identified in LRP1 and F8 genes
Nucleotide change Amino acidchange Gene Region NCBI Code Prevalence of mutantallele in the NCBI (%)* Classification Prevalence of mutant allele inthe study Patients/Controls % (N) P* C 2767 T Arg 767 Arg LRP1 Exon 14 rs145618022 0.2 Mutation T: 0.67 (1)/0.68 (1) 1
C 2805 T Thr 780 Ile LRP1 Exon 14 rs34492744 0.3 Mutation T: ZERO (0)/0.68 (1) 1
C 28701167 T NA LRP1 Intron 14 rs35306126 0.6 Mutation T: ZERO (0)/0.68 (1) 1
C 28701197 T NA LRP1 Intron 14 rs34709055 0.6 Mutation T: ZERO (0)/0.68 (1) 1
G 2996126 A NA LRP1 Intron 15 rs138196021 0.1 Mutation A: 0.67 (1)/ZERO (0) 1
C 3137154 T NA LRP1 Intron 16 rs181881519 0.1 Mutation T: 0.67 (1)/ZERO (0) 1
G 3137181 A NA LRP1 Intron 16 rs1800174 40.0 Polymorphism A: 39.3 (59)/35.8 (53) 0.59
G 31371111 A NA LRP1 Intron 16 Novel NA Mutation A: 0.67 (1)/ZERO (0) 1
G 3264 -172 A NA LRP1 Intron 17 Novel NA Mutation A: ZERO (0)/0.68 (1) 1
C 3264 - 3 T NA LRP1 Intron 17 Novel NA Mutation T: 0.67 (1)/ZERO (0) 1
G 3376 A Ser 970 Ser LRP1 Exon 18 rs78054559 0.1 Mutation A: 0.67 (1)/ZERO (0) 1
T 33801114 C NA LRP1 Intron 18 rs34474417 2.2 Polymorphism C: 2.66 (4)/ZERO (0) 0.13
T 1444 - 22 C NA F8 Intron 9 rs5986899 0.7 Mutation C: 0.67 (1)/ZERO (0) 1
C 7053 1 32 T NA F8 39 UTR rs5986887 0.9 Mutation T: 0.67 (1)/ZERO (0) 1
This table shows information about mutations and polymorphisms found in the study, including their prevalence in patients and controls, and NCBI database.
*Genetic alterations previously described were classified as mutation (allelic prevalence , 1%) or polymorphism (allelic prevalence $ 1%) according to their prevalence in the population of NCBI analysis, composed of 1000 healthy individuals (2000 alleles).
**P values were calculated by the Fisher’s exact test. Abbreviations: NCBI 5 The National Center for Biotechnology Information; Arg 5 Arginine; Thr 5 threonine; Ile 5 Isoleucine; Ser 5 Serine; 39 UTR 5 39 untranslated region; NA 5 Not Applicable.
FVIII, and the complex VWF/FVIII is crucial for the survival of
FVIII in the blood circulation by protects it against impropriated
proteolytic activation and premature clearance. Therefore, it is not
surprising that plasma VWF level plays a critical role in regulating
plasma FVIII levels. However, the mechanisms underlying the
inter-individual variability of plasma FVIII levels are not yet fully
under-stood, and these main determinants explain only a part of FVIII
levels variation. Thus, it has been suggested that others unknown
important factors (mainly genetic factors) could have participation
on FVIII variation
34.
Previous studies that evaluated patients with VTE and increased
FVIII levels suggested a familial clustering of high FVIII levels
12–14.
For example, in a retrospective study of 177 VTE patients with high
FVIII levels, Bank et al. (2005) observed that 40% of their first-degree
relatives also presented FVIII levels above the 75th percentile of the
normal population. Moreover, these first-degree relatives with
ele-vated plasma FVIII levels also demonstrated increased risk for
VTE
13.
In the present study, we hypothesized that genetic variations in
gene regions coding the affinity sites between FVIII and LRP1 might
influence plasma FVIII levels, and that a cohort of VTE patients with
persistently elevated FVIII levels might be an interesting target for
screening these genetic variations. By selecting this population, we
hoped increase the possibility for detecting clinically relevant
muta-tions or polymorphisms associated with elevated FVIII levels. With
this strategy, we identified two mutations in the analyzed regions of
F8 gene, one in intron 9 and other in the 39 UTR region, present in
one VTE patient. Morange et al. (2005) evaluated these same regions
of F8 gene, in 10 healthy individuals with the top higher and lower
FVIII levels of 424 healthy individuals. No polymorphism was
detected in these regions of the F8 gene
14. In accordance with our
results, genetic variations in these F8 gene regions are probably not
associated with elevated FVIII levels.
Two studies simultaneously identified the LRP1 as a first
candid-ate clearance receptor for FVIII
20,21, and in the last decade, several
studies have analyzed the influence of LRP1 on FVIII levels in
humans
14,25–28. In particular, investigators have focused their
research on analyze the association between LRP1 polymorphisms
and plasma FVIII levels, and VTE risk. Association between LRP1
D2080N and –25C/G polymorphisms with decreased FVIII
levels
14,26, and LRP1 C663T polymorphism with increased FVIII
levels and with a three-fold increased risk of VTE have been
described
25.
Although we identified 12 mutations/polymorphisms in the
region of LRP1 gene, these were not associated with FVIII levels.
Our results suggest no association of genetic variations located in
the first six CRs of cluster II of LRP1 with plasma FVIII levels, but we
could not ruled out the role of variations affecting other important
affinity region, localized in cluster IV
27–29. We can also consider that
despite the efficiency which LRP1 binds FVIII and mediates its
intra-cellular degradation, the absence of LRP1 resulted only in a partial
inhibition of FVIII degradation when evaluated in vivo assays,
indi-cating that other LRP1-independent pathways are involved in FVIII
uptake
22,35.
Finally, in this study, we showed that patients with a median of 10
years after the first episode of VTE presented higher FVIII levels
when compared to healthy controls, but the magnitude of this
dif-ference was significantly lower than that observed at an earlier time
point. These findings suggest the presence of stimulus to increase
FVIII levels that could be stronger during the initial years after the
VTE episode. CRP is expressed at low levels in the absence of an acute
inflammatory stimulus, and the results found in our patients
sug-gested that, they did not present an acute inflammatory response in
both assessments. Our results corroborate other studies that analyzed
inflammatory markers after VTE and almost all demonstrated
decline of CRP after a short time
11,36,37Thus, these findings suggested
that increased FVIII levels observed in patients with previous VTE
be, in part, consequence of a sub-clinical chronic inflammation, not
revealed by CRP. In accordance with this hypothesis, Bouman et al.
(2014) performed an evaluation 63 months after the acute
throm-botic episode describing increased levels of IL-6 in patients when
compared to controls
38. Thus, we cannot rule out that even years
after the thrombotic episode, a sub-clinical chronic inflammatory
state could contribute to high levels of FVIII in VTE patients.
Thus far, strengths of the present study are the evaluation of
spe-cific gene regions coding the affinity sites between FVIII and LRP1
that might influence plasma FVIII levels, in a well-defined
popu-lation of cases with a history of high FVIII levels and matched
con-trols. However, an important limitation of this study is the relatively
small sample size, which should be considered in the evaluation of
associations between the genetic alterations and high FVIII levels.
Therefore, this was an exploratory study and a relationship between
the genetic variations found, high FVIII levels and VTE remains to be
clarified by larger studies.
Conclusions
We demonstrated a persistent increase of plasma FVIII levels in a
subset of patients with VTE, but in a much lower magnitude after 10
years of VTE episode when compared to the first evaluation 3-years
after the episode. Moreover, we observed no relationship between
genetic variations in the gene regions coding sites of affinity between
LRP1 and FVIII proteins and FVIII levels.
Methods
Study Population.Written informed consent was obtained from all participants, and the study was approved by the Ethics Committee of University of Campinas. All the methods were carried out in accordance with the approved guidelines. This was a case-control study and initial cohort consisted of 314 adult patients with a first episode of VTE, between January 1990 and September 2004, followed up at the Hemostasis and Thrombosis Clinic, at University of Campinas, Brazil. Patients with cancer, liver, renal, or systemic inflammatory diseases were excluded. VTE was confirmed by imaging tests. In a first assessment, with a median time of three years
Table 3 | Plasma FVIII levels according to genotypes in VTE patients
Polymorphisms N FVIII - 1u Assessment [IU/dL]* FVIII - 2u Assessment [IU/dL]*
G 3137181A GG 27 238.3 (219.3–276.3) 164.0 (153.3–174.5) GA 37 252.6 (223.6–326.0) 156.0 (150.5–172.8) AA 11 240.8 (223.5–283.0) 158.0 (134.0–160.2) P 5 0.66** P 5 0.47** T 33801114 C TT 71 239.9 (218.3–298.1) 157.1 (148.0–171.0) TC 4 233.4 (212.7–241.7) 168.0 (163.0–173.0) P 5 0.43** P 5 0.33**
This table shows plasma FVIII levels in VTE patients according their genotypes for LRP1 polymorphisms. *Results are expressed as median (interquartile range).
after the acute episode, FVIII levels were evaluated in all 314 patients and in 314 matched controls, without a medical history of VTE from the same geographic region and ethnic background of the patients. Seven years later, all 104 patients from this initial cohort who originally presented FVIII levels above the 90thpercentile (P90)
were recruited for a second assessment, but only 75 patients (72.1%) agreed to participate. Seventy-four healthy controls were again selected according to age, gender, ABO blood group, geographic region, ethnic background, and the same exclusion criteria were used for the study entry.
Laboratory Methods.After an overnight fast, venous blood samples (18 mL) were collected from all participants, from the antecubital vein into into VacuetteH tubes (Greiner Bio-One, Austria): 0.129 mmol/L trisodium citrate tube,
ethylenediaminetetraacetic (EDTA) tube, and Z Serum Sep Clot Activator tube. Samples were immediately centrifuged for 20 minutes at 1500 g, and plasma/serum were immediately frozen and stored at 280uC.
FVIII Activity.FVIII activity levels were measured by a one-stage clotting assay with FVIII-deficient plasma (Siemens, Marburg, Germany) as recommended by the manufacturer. FVIII were determined in duplicate on automated coagulation analyzer (BCS XP, Siemens, Marburg, Germany). This methodology was performed in both assessments. Normal range was 62 to 151 IU/dL.
C-reactive protein.Serum high sensitive C-Reactive protein (hs-CRP) levels were determined by a nephelometric method (Siemens, Marburg, Germany), on Siemens BN ProSpec analyzer. Normal range was , 0.50 mg/dL.
ABO blood group.ABO blood group was determined by agglutination and adsorption-elution test.
Sequencing of the LRP1 and F8 gene regions potentially involved in LRP1-FVIII binding.Three regions were selected in F8 gene: (i) exons 10 and 11 (containing the site Arg484-Phe509 in the A2 domain), (ii) exon 16 (containing the site Lys1804-Ala1834 in the A3 domain), and (iii) exon 26 (containing the site Thr2303-Tyr2333 in the C2 region). In the LRP1 gene, exons 14 to 20 (containing the site of the first six CRs of cluster II) were selected. All these regions were amplified by polymerase chain reaction (PCR). Primer sequences, PCR products and length of PCR products are listed in the Supplementary Table S1.
PCR products were checked on 2% agarose gels (Uniscience, Brazil) stained with 2 ug/ml ethidium bromide (Invitrogen, USA), and purified by commercial kit GFX PCR DNA and Gel Band Purification Kit (GE Healthcare). Subsequently, these products were sequenced by the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Life Technologies, USA) according to manufacturer’s instructions for subsequent electrophoresis in automatic sequencer ABI PRISMH 3500 Genetic Analyzer (Applied Biosystems, USA). The analysis of sequencing was performed by the Chromas software (version 2.3.0), comparing the samples sequences to database of The National Center for Biotechnology Information (NCBI), NG_016444.1 for LRP1 gene and NG_011403.1 for F8 gene.
Statistical analysis.Continuous variables were described as median and interquartile range. Medians between patients and controls were compared by the Mann-Whitney test, and categorical variables were compared by the Fisher’s exact test. Genotype frequencies between patients and controls were compared by the Fisher’s exact test. Medians of FVIII levels according the genotypes were compared by Mann-Whitney or Kruskal-Wallis tests. A P value ,0.05 was considered statistically significant and all tests were two-tailed. All analysis was performed by the R Foundation for Statistical Computing, version 3.0.1.
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Acknowledgments
The authors would like to thank Fundaça˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) for the financial support (FAPESP, nu 2009/53543-6).
Author contributions
L.F.B., L.H.S., F.L.O., E.V.P. and J.M.A-B. designed the experiments and analyzed the data. L.F.B., E.V.P. and J.M.A.-B. wrote the manuscript text. L.F.B. performed the experiments and L.H.S. assisted with primers design. All authors reviewed the manuscript.
Additional information
Supplementary informationaccompanies this paper at http://www.nature.com/ scientificreports
Competing financial interests:The authors declare no competing financial interests. How to cite this article:Bittar, L.F., Siqueira, L.H., Orsi, F.L.A., De Paula, E.V. & Annichino-Bizzacchi, J.M. Genetic variations in sites of affinity between FVIII and LRP1 are not associated with high FVIII levels in venous thromboembolism. Sci. Rep. 5, 9246; DOI:10.1038/srep09246 (2015).
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